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David L. Rimm M.D., Ph.D
Professor, Director of Translational Pathology
Dept. of Pathology
Yale University School of Medicine
Beyond Brown;
Immunohistochemistry in
the Future
Disclosure
• I am a consultant to Genoptix/Novartis,
Applied Cellular Diagnostics, BMS,
Amgen, Optrascan, Biocept, Perkin Elmer,
and Metamark Genetics
• I hold equity in Metamark Genetics
• Cepheid, Genoptix. Gilead
Pharmaceuticals Kolltan and OncoplexDx
fund research in my lab.
The value of two dimensional
information: Morphology
Auguste Renoir :
The Luncheon of the Boating Party
C.1881
Claude Monet:
The Stroll, Camille Monet and Her Son
Jean (Woman with a Parasol)
C. 1875
Immunoperoxidase stain
Uses of IPOX:
•Identification (binary)
•Reading/estimation (ordinal)
•Quantification (continuous)
The human eye is not a great tool for
assessment of intensity
A Long History of Quantitative
Microscopy• Equipment for quantitative morphology
dating back to the 1960’s
• The CAS 200 (Jim and Sarah Bacus)
• Chromavision ACIS
• Bacus Labs, BLISS system
• Compucyte, Applied Imaging,
Areol/Genetix, Definiens, CRI INform
• Aperio and Bioimagene
Commercially Available
Pathology-Focused Image
Analysis Platforms• Definiens
• Visiopharm
• Perkin Elmer Vectra/Mantra/INform
• Hamamatsu Nanozoomer
• Leica/Aperio
• Tissuegnostics
• Ventana (formerly Bioimagene)
• Optra SCAN
• GE MultiOmyx
• Genoptix (formerly HistoRx) AQUA
• Many others sold as research platforms
AQUA®: objective analyte measurement on a
tissue slide based on co-localization
Step 1: Mask (define region of interest, exclude stroma, blank space, etc) =
colocalization with Cytokeratin for carcinoma
Step 2: Define the numerator (target) and denominator (compartment)
Concentration =Numerator
Denominator Σ compartment
pixel area
Σ target intensity
in compartment pixels
Step 3: Calculate the AQUA score
Step 4: Convert to absolute concentration or normalize to set
of uniform standards
= AQUA
score
TMA
WTS
TMA-Tissue Microarray
WTS-Whole Tissue Section
Cytokeratin Tumor Mask
Combine DAPI image and
cytokeratin image then cluster to
assign each pixel to a subcellular
compartment
Estrogen Receptor
Σ compartment
pixel area
Σ target intensity
in compartment pixels= AQUA
score
Generating
the AQUA®
score
Development and Commercialization Of a
Quantitative Protein Measurement Technology
(AQUA) from the lab to the patient
Precision Results (ER-alpha)
Pearson R Slope
Day 1 v. Day 2 .97 .97
Day 1 v. Day 3 .97 1.01
Day 2 v. Day 3 .98 1.04
%CV = 4.2
Mark Gustavson and
Jason Christiansen
Alley Welsh
Standardized Index Array ER antibody used is 1D5
Lowest positive vs. highest negative
ER
ER Alley Welsh
Expanded “levels” to visualize thresholdcontracted dynamic range of grayscale (max RGB input level 25516)
ER ER
ER ER
Discordant classification of ER status in YTMA 130 cohort
Welsh et al, JCO 2011
Annotation
Algorithm Markup
Quantification of DAB by
Aperio Pixel Counter
y = 0.9809xR² = 0.9948
0
5000
10000
15000
0 5000 10000 15000
Nu
cle
ar
AQ
UA
Sco
re (
User
2)
Nuclear AQUA Score (User 1)
y = 0.9086xR² = 0.9629
0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40 50 60 70 80 90 100
Perc
en
t P
osit
ive N
ucle
i (U
ser
2)
Percent Positive Nuclei (User 1)
y = 0.9211xR² = 0.914
0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40 50 60 70 80 90 100
Perc
en
t P
osit
ive N
ucle
i (P
ath
. 2)
Percent Positive Nuclei (Path. 1)
Comparison
Between
Methods
(reproducibility)Pathologist vs
Pathologist
DAB semi vs DAB
semi
QIF (AQUA) vs
QIF (AQUA)
Elizabeth Zarrella and Veronique Neumeister
0
20
40
60
80
100
0 20 40 60 80 100
Perc
en
t P
os.
Nu
cle
i (A
peri
o)
Percent Pos. Nuclei (Pathologist)
0
2000
4000
6000
8000
10000
12000
14000
0 20 40 60 80 100
Nu
cle
ar
AQ
UA
Sco
re
Percent Pos. Nuclei (Pathologist)
0
2000
4000
6000
8000
10000
12000
14000
0 20 40 60 80 100
Nu
cle
ar
AQ
UA
Sco
re
Percent Positive Nuclei (Aperio)
Comparison
Between
Methods
Pathologist vs
DAB semi
DAB semi vs
QIF(AQUA)
Pathologist vs
QIF(AQUA)
Elizabeth Zarrella and Veronique Neumeister
Although there was a high proportion of positive staining tumor cells in all of
the 3+ samples the range of staining intensity varied.
2+ 3+ 3+ 3+ 3+
Different Intensities of HER2 IHC Staining Observed Within HER2(+) Patient
Population
Intensities of HER2 IHC Staining Observed in HER2+ Patients From
the HERA Trial
Zabaglo L, et al. Presented at 33rd SABCS; December 8-12, 2010; San Antonio, TX. Abstract PD10-01.
Slide provided by Mitch Dowsett
Regression of IHC vs QIF scores for
CB11 and SP3 in YTMA 263
CB11 SP3
Daniel Carvajal
Scoring Affects Outcome Prediction
y = 0.0173x + 8.3595R² = 0.1099
0
20
40
60
80
100
120
140
160
0 1000 2000 3000 4000 5000 6000
mem
bra
ne H
sco
res
cMet AQUA scores
Correlation cMet AQUA (Ventana) and DAB based IHC outside scoring by eye
y = 0.0234x + 32.376R² = 0.1308
0
20
40
60
80
100
120
140
160
180
0 1000 2000 3000 4000 5000 6000
mem
bra
ne c
yto
pla
sm
ic H
-sco
r
Correlation cMet AQUA (Ventana) and DAB based IHC outside scoring by eye
K-M plots of Met scored by
AQUA
P=0.012
The Goal: To Measure Protein on a slide
with the accuracy of Analytical Chemistry
LOD= limit of detection
LOQ = limit of quantification
LOL = limit of linearity
S/N = 10
S/N = 3
Outline for Presentation
• Domain Specific Assessment of HER2 in
Breast Cancer
• Quantitative Multiplexed assessment of
TILs
• Beyond QIF
T-DM1 neratinib
SP3
CB11
A0485
(Herceptest®)
Antibody
Binding Sites
D8F12
4B5
Pert.
What are we using for IHC?
Antibody N Percent
4B5 568 55%
A0485 54 5%
CB11 24 2%
Herceptest 296 29%
SP3 52 5%
other 37 4%
Total 1031
Studies done
“by eye”
cannot see
discordance
Less than 1%
discordance between
cytoplasmic and
extracellular domain
antibodies
SP3 C vs CB11 C (NCCTG 9831 Arm C)
Data from NCCTG9831 from ongoing collaboration with Edith Perez and Karla Ballman
Images
SP3
21 262 78
CB11
DAB shows same effect, but
more subtle
CB11 (ICD) SP3 (ECD)
HER2 Standardization Array
(YTMA 263)3
+, A
MP
1+,
NO
T A
MP
0, N
OT
AM
P
2+,
AM
P
3+,
AM
P
1+,
NO
T A
MP
0, N
OT
AM
P
2+,
NO
T A
MP
CEL
L LI
NES
NO
RM
AL
BR
EAST
TI
SSU
E (N
BT)
1 2 3 4 5 6 7 8 9 10
2008 2011 2011 2010 2005 2009 2004 2011 MB453 NBT
2007 2011 2011 2007 2004 2009 2004 2011 BT20 NBT
2011 2011 2011 2006 2004 2008 2009 2011 MCF7 NBT
2011 2011 2011 1998 2003 2007 2009 2011 MB468 NBT
2011 2011 2010 2004 2003 2007 2009 2011 ZR751 NBT
2010 2011 2010 2004 2002 2006 2007 2011 MB361 NBT
2009 2011 2007 2003 2002 2011 2007 2011 SKBR3 NBT
2009 2011 2007 2003 2002 2011 2011 2011 UACC812 NBT
2008 2010 2006 2011 2002 2011 2011 2007 MDA231 NBT
2008 2010 2006 2010 2002 2011 2011 2007 BT474 NBT
HER2
CB11
Serial section Reproducibility for
Domain Specific antibodies
Extra-Cellular Domain (SP3) Intra-Cellular Domain (CB11)
Daniel Carvajal
1:1 line
Daniel Carvajal
Cut-point is around
500
Daniel Carvajal
Cut-point is around
1500
Daniel Carvajal
HER2 CB11 analysis
Daniel Carvajal
Using Joinpoint to Assess Ab Sensitivity and
Specificity to Predict FISH amplification in
YTMA 263
HER2
antibody
Joinpoint
cut-point
(AU)
Sensitivity
(%)
Specificity
(%)
Positive
predictive
value (%)
Negative
predictive
value (%)
CB11 1299.3 94.12 85.48 78.05 96.36
A0485 1495.9 90.62 85.71 78.38 94.12
SP3 525.8 88.24 98.41 96.77 93.94
D8F12 767.6 90.91 93.88 90.91 93.88
Daniel Carvajal
Using Spectrum/Aperio scores from
Membrane/Total pixel count for HER2 CB11
Daniel Carvajal
Using Joinpoint to Assess Ab Sensitivity and
Specificity to Predict FISH amplification in
YTMA 263 (Chromogenic IHC)
HER2
antibody
Joinpoint
cut-point
(AU)
Sensitivity
(%)
Specificity
(%)
Positive
predictive
value (%)
Negative
predictive
value (%)
CB11 71.65 96.3 64.29 63.41 96.43
SP3 75.75 89.66 82.61 76.47 92.68
Daniel Carvajal
Conclusions
• The binding site of the HER2 antibody
effects the results of the HER2 assay
• Compared to FISH, the cytoplasmic
domain is more sensitive for prediction of
amplification, while the extracellular
domain is more specific.
• This result is independent of detection
method (QIF vs DAB)
• Prediction of response to therapy is more
important than prediction of FISH
amplification
HeCOG 10/05 Trial arms• Group A epirubicin (110 mg/m2) followed by paclitaxel (200 mg/m2)
CMF (cyclophosphamide; 840 mg/m2, methotrexate; 57 mg/m2 and
fluorouracil; 840 mg/m2) every 2 weeks.
• Group B epirubicin (110 mg/m2) followed CMF every 2 weeks
followed later by Docetaxel; 35 mg/m2.
• Group C epirubicin (110 mg/m2) followed by CMF followed later by
Paclitaxel 80 mg/m2.
• HER2 positive patients treated with trastuzumab every 3 weeks
for 1 year after the completion of chemotherapy. In these
patients hormonal treatment with tamoxifen or anastrazole will
be started following termination of treatment with trastuzumab.
In summary, all patients received chemotherapy and the HER2 positive
were further treated with sequential trastuzumab (like arm B of N9831).
HeCOG 10/05 patient subgroups
Table 2. Biological subgroups
Subgroup N (%)
ER(+)/PgR(+)/HER2(-) 366 (51.9)
ER(+)/PgR(+)/HER2(+) 90 (12.8)
ER(+)/PgR(-)/HER2(-) 66 (9.4)
ER(+)/PgR(-)/HER2(+) 23 (3.3)
ER(-)/PgR(-)/HER2(-) (TNBC) 68 (9.6)
ER(-)/PgR(-)/HER2(+) 69 (9.8)
ER(-)/PgR(+)/HER2(-) 15 (2.1)
ER(-)/PgR(+)/HER2(+) 7 (0.9)
Not determined 1 (0.1)
Table 3. Treatment subgroups
Treatment group N (%)
ER(+) with hormonotherapy 527 (96.7 of ER[+])
ER(+) without hormonotherapy 18 (3.3 of ER[+])
HER2(+) with trastuzumab 186 (98.4 of HER2[+])
HER2(+) without trastuzumab 3 (1.6 of HER2[+])
Hormonotherapy and trastuzumab 111 (15.7 of total)
ER(-) with hormonotherapy 0 (0)
HER2 (-) with trastuzumab 0 (0)
HER2 ICD/ECD protein expression by quadrants
Q1 Q2
Q3
Q4
• Cut-points obtained
using YTMA 263 slides
stained alongside
HeCOG and analyzed
using Joinpoint
software.
• Quadrants:
- Q1 (ICDlow/ECDhigh):
5/159 patients (3.1%).
- Q2 (ICDhigh/ECDhigh):
59/159 patients (37.1%).
- Q3 (ICDhigh/ECDlow):
24/159 patients (15.1%).
- Q4 (ICDlow/ECDlow):
71/159 patients (44.7%).
Outline for Presentation
• Domain Specific Assessment of HER2 in
Breast Cancer
• Quantitative Multiplexed assessment of
TILs
• Beyond QIF
TILs in Breast CancerSalgado et al – Annals of Oncology
The Yale Immunoscore: Triple Multiplexing
to measure CD3, 8 and 20 with quantitative
immunofluorescence (AQUA) • Quantitatively assess each marker in the stroma
• Validate on Tonsil compared to flow cytometry and then validate on
breast, lung and colon cohorts
• Measure CD8 (or in the future CD4 or other markers) in the CD 3
compartment
• Could be done on whole slides or TMAs
• Schematic:CD3 1:100 (Novus,
Rabbit Monoclonal)
CD8 1:250 (Dako,
Mouse Monoclonal)
CD20 1:150 (Dako,
Mouse Monoclonal)
Rabbit Envision
Secondary
Mouse IgG1
Secondary HRP
(eBioscience)
1:100
Mouse IgG2a
Secondary HRP
(AbCam) 1:200
Biotinylated
Tyramide
Alexa 750-
Streptavidin
Fluoroscein/
Tyramide
Cy5/Tyramide
Rabbit
Cytokeratin
1:100 (Dako)
Goat Anti-Rabbit
Alexa 546
DAPI Jason Brown
DAPI CK CD20
CD3
CD8
Human Tonsil
Hematoxylin-Eosin
Flow Cytometry
Analysis on Tonsil
Specimens
Jason Brown
Measurement by QIF is very similar to measurement by
flow cytometry of tonsil control tissue
0
10
20
30
40
50
60
70
80
90
100
Sample 1 Sample2 Sample3 Sample 4
% Lymphocyte Comparison
Flow Cytometry
QuantitativeImmunofluoresence
0
10
20
30
40
50
60
70
80
90
100
Sample1 Sample 2 Sample 3 Sample 4
% CD3 Comparison
Flow Cytometry
QuantitativeImmunofluoresence
0
10
20
30
40
50
60
70
80
90
100
Sample 1 Sample 2 Sample 3 Sample 4
% CD20 Comparison
Flow Cytometry
QuantitativeImmunofluoresence
0
10
20
30
40
50
60
70
80
90
100
Sample 1 Sample 2 Sample 3 Sample 4
% CD8 Comparison
Flow Cytometry
QuantiativeImmunofluoresence
Jason Brown
Comparison between AQUA and Pathologist on
Yale Breast Cancer Cohort 2
CD3 CD8
CD20
Jason Brown and Kurt Schalper
Jason Brown and Kurt Schalper
Measuring FOVs in whole sections of breast biopsies
Jason Brown and Kurt Schalper
Quantitative TILS predict
PathCR in Neoadjuvant Cohort
TILs show Prognostic Value in Breast Cancer
Distribution of TILs signal in NSCLC samples on YTMA79
Jason Brown and Kurt Schalper
OS by CD3 Signal on YTMA79
P=0.03
100 93 74 56 49 34 27
100 83 65 41 37 28 23
OS by CD8 Signal on YTMA79
P=0.007
100 91 72 52 46 32 25
100 85 67 44 39 30 25
OS by CD20 Signal on YTMA79
P=0.06
100 89 72 58 51 32 25
100 88 67 39 35 30 25
100 91 72 52 46 32 25
100 85 67 44 39 30 25
OS by CD8 in CD3 Signal on YTMA79
P=0.02
Outline for Presentation
• Domain Specific Assessment of HER2 in
Breast Cancer
• Quantitative Multiplexed assessment of
TILs
• Beyond QIF
30-site
chelating
polymer
x 6 polymers
= 180 atoms
per antibody
Labelling antibodies for
CyTOF or MSIHC
Isotopically
enriched
lanthanide
ions (+3)
Secondary Ion Mass Spec
compared to Fluorescence
Why is mass spectrometry better suited for multiplexing
compared to fluorescence?
Angelo et al.
Nolan Lab, Stanford University
Multiplexed Ion Beam Imaging
Angelo et al.
Nolan Lab, Stanford University
Multiplexed Ion Beam Imaging
Angelo et al.
Nolan Lab, Stanford University
Multiplexed Ion Beam Imaging
10-color imaging of human breast tumors using MIBI
Angelo et al.
Nolan Lab, Stanford University
March 2014
Thanks to:Rimm Group:
Kurt Schalper
Daniel Carvajal
Jason Brown
Susan Combs
James Smithy
Lauren Moore
Joe McLaughlin
Mehmet Altan
John McGuire
Joanna Hu
Vasiliki Pelakanou
Mengyao Feng
Nikita Mani
Yan Song
Maria Toki
Nick Goel
Yale Pathology Tissue Services
Lori Charette
Sudha Kumar
Veronique Neumeister
Yalai Bai
(Google YPTS)
Outside Yale Collaborators
David Hicks (U Rochester)
Edith Perez and NCCTG
George Fountzilas – HeCOG
Amanda Psyrri – HeCOG
Konstantine Kalogeras -HeCOG
Yale Collaborators
Lajos Pusztai
Yuval Kluger
Christos Hatzis
Work supported by grants from the NCI, DOD, BCRF, and SRAs from Gilead
Sciences and Genoptix
Rimm Lab Alums:Vamsi Velchetti
Elsa Anagnostou
Anastasios Dimou
Alley Welsh
Robert Camp
Maria Vassilikapoulou
Huan Cheng
Jennifer Bordeaux
Vamsi Velchetti
Elizabeth Zarrella
Hallie Wimberly
www.tissuearray.orgRimm Lab, Fall 2014